This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Sir2 (Silent Information Regulator-2) proteins function in aging and lifespan regulation in multiple model organisms. Preliminary work indicates that one mammalian Sir2 protein, SIRT6, suppresses genomic instability and attenuates the onset of several age-associated pathologies. However, the molecular mechanisms of SIRT6 function are not known. Thus, a systematic characterization of the basic molecular mechanisms through which SIRT6 functions in human cells should be instrumental for elucidating fundamental biological processes that impact on human health and aging. A series of biochemical, cellular, and global proteomic and genomic analyses will be carried out to define the molecular functions of SIRT6 in human cells. We propose: 1. To elucidate the molecular mechanisms by which SIRT6 functions in chromatin regulation. Post-translational modifications of histones at chromatin play important roles in gene expression programs and DNA damage responses. Preliminary results indicate that SIRT6 is tightly associated with, and may catalyze modifications of, histones at chromatin. We will define the enzymatic activity of SIRT6 at chromatin and the effects of altered SIRT6 levels on chromatin structure and functional states. 2. To elucidate novel SIRT6 regulated molecular pathways. Proteomic and biochemical approaches will be taken to isolate and identify SIRT6 substrates, protein binding partners, and macromolecular complexes. In addition, genome wide approaches will be employed to identify SIRT6-regulated genes and SIRT6 binding sites at chromatin. Mass Spectrometry analysis in collaboration with the UCSF Mass Spectrometry Facility will be essential for these aims, in the identification of post-translational modifications on histones and other proteins catalyzed by SIRT6, and of SIRT6 interacting factors and macromolecular complexes.